First Report of Shoot Blight on Cinnamomum cassia Caused by Neofusicoccum parvum in China

Abstract

Cinnamomum cassia Presl, an aromatic tree of the Lauraceae family, is widely cultivated in southern China as a source plant for traditional medicine and spice (Zhang et al. 2019). In June 2025, a shoot blight was observed on C. cassia plants in a plantation at the Guangxi Botanical Garden of Medicinal Plants, with an incidence rate of 60% based on a survey of 120 plants. The disease attacks tender shoots, causing leaf curling, wilting, and local dieback. To identify the causal agent, 15 pieces (3×3 mm) of diseased apical bud tissue collected from 5 individual plants were surface-sterilized (75% ethanol for 30 s, 2.5% NaClO for 90 s, rinsed three times in sterile distilled water), placed on potato dextrose agar (PDA), and incubated at 28°C for 3 days. Hyphal tips were transferred to fresh PDA for pure cultures. Of 33 isolates obtained, 27 shared similar morphology. The colonies grew relatively fast: initially white aerial mycelium, turning gray at the center after 4 days, then entirely gray-black. No spore production was observed even after 45 days on PDA, water agar, or carrot medium, including at inoculation sites on young shoots. The remaining six isolates exhibited different colony morphologies and were not pathogenic in preliminary tests (see below). Three isolates (RGK, RGK2, RGK3) were selected for molecular identification. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1), and beta-tubulin (TUB2) regions were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences obtained from the three isolates were found to be 100% identical to each other. Consequently, only the sequences of strain RGK were deposited in GenBank, with accession numbers PX888074 (ITS), PZ281588 (TEF1), and PX899692 (TUB2). BLAST analysis revealed that the ITS, TEF1, and TUB2 sequences of isolate RGK showed 99.65% (570/572 bp), 99.66% (292/293 bp), and 100% (436/436 bp) similarity to the corresponding sequences of Neofusicoccum parvum (accession nos. MW404560, MN175952, and EU673095, respectively). Phylogenetic analysis was based on a concatenation of ITS, TEF1, and TUB2 nucleotide sequences using MEGA 11 software and placed RGK within the N. parvum clade. Based on morphological characteristics, BLAST and phylogenetic analysis, isolate RGK was identified as N. parvum. Pathogenicity was tested using the mycelial fragment suspension (Donzelli and Churchill 2007). A total of nine isolates (three representative isolates RGK, RGK2, RGK3, plus the six morphologically distinct ones) were inoculated on detached young shoots. The isolates were grown on cellophane-overlaid plates. Mycelia were scraped off with a sterile spatula and suspended in sterile distilled water to achieve a concentration of 20 mg/ml based on fresh weight. Each detached young shoot was sprayed with 10 ml of the suspension, placed in a Petri dish with moist sterile filter paper, and incubated at 28°C with 80% relative humidity. Six replicates were used per treatment. Control shoots received 10 ml of sterile distilled water. After 6 days, only RGK, RGK2, and RGK3 caused characteristic black lesions, whereas the other six produced no symptoms. Controls remained asymptomatic. The fungus reisolated from the diseased shoots showed identical colony morphology and ITS sequence to the original isolates, fulfilling Koch's postulates. N. parvum has been reported to cause dieback on Cinnamomum daphnoides (Hao et al. 2026). To our knowledge, this is the first report of N. parvum causing shoot blight on C. cassia in China. This finding provides a reference for the prevention and control of this disease on C. cassia.